Abstract LB101: Novel Drug-responsive domain (DRD)-based regulation technology enables tightly controlled activity of potent membrane-bound IL12 in adoptive cell therapies
Authors: Sean Gregory Smith, James A. Storer, Dexue Sun, Dan Jun Li, Benjamin Primack, Theresa Ross, Scott LaJoie, Jeremy Tchaicha, Dhruv Sethi, Jan ter Meulen, Michelle Ols
Journal: Cancer Research (2022)

Abstract Interleukin 12 (IL12) is an attractive cancer immunotherapeutic known to be extremely potent against solid tumors preclinically. However, the clinical utility of IL12 has been limited by toxicities stemming from high systemic cytokine exposure. Thus, armoring cellular therapies such as chimeric antigen receptor T cells (CAR-Ts) or tumor infiltrating lymphocytes (TILs) with IL12 will require technologies that provide tight control of IL12 expression and localization. Herein, we describe a tightly regulated version of IL12 for use in cellular therapies. First, we show that tethering IL12 to the membrane increases the activity of IL12 at the tumor site in vivo while reducing potential systemic toxicities. Membrane bound (mbIL12) was compared to secreted IL12 in the syngeneic CD8 gp100 TCR transgenic PMEL model. In this model, PMEL T cells transduced with mbIL12 demonstrated similar reduction in B16 tumor outgrowth as secreted IL12. Like secreted IL12, mbIL12 enhanced the expansion of PMEL T cells and retained T cell extrinsic activities, such as activation of myeloid cells and remodeling the tumor microenvironment. However, mbIL12 showed reduced toxicity signals as compared to secreted IL12, including a reduction in serum IFNy. Likewise, in a xenograft system, CD19-CARTs expressing human mbIL12 demonstrated enhanced potency against Raji tumors. To enhance the safety of mbIL12 further we sought to regulate its expression using Obsidian’s cytoDRIVE® technology. This platform consists of small, fully human protein sequences called drug responsive domains (DRD)s, such as carbonic anhydrase 2, that enable regulation of expression of a fused target protein under the control of FDA-approved, orally bioavailable small molecule ligands, such as, acetazolamide. In the absence of ligand (the “off-state”), the fusion protein is unfolded and degraded. In the presence of ligand (the “on-state”), the DRD is stabilized, allowing for protein expression and function. Thus, the cytoDRiVE® system acts as a titratable and reversible rheostat for on-demand protein activity. While a single DRD can enable some regulation of mbIL12 levels, we found that adding a modulation hub that increases the multiplicity of DRDs greatly improves regulation. Indeed, these modifications enabled off-state levels of mbIL12 that were indistinguishable from untransduced controls in HEK and Jurkat cell lines as well as primary human CD19 CAR-T-cells using flow cytometry. These modifications also enhanced mbIL12 regulation, leading up to a 30-fold dynamic range of mbIL12 abundance. Regulated mbIL12 was demonstrated to be active by phosphorylation of STAT4 in bystander NK cells in vitro. Combining DRDs with modulation hubs and membrane tethering enables the control of highly potent, previously clinically intractable cytokines, such as IL12, for use in enhancing cell therapies. Citation Format: Sean Gregory Smith, James A. Storer, Dexue Sun, Dan Jun Li, Benjamin Primack, Theresa Ross, Scott LaJoie, Jeremy Tchaicha, Dhruv Sethi, Jan ter Meulen, Michelle Ols. Novel Drug-responsive domain (DRD)-based regulation technology enables tightly controlled activity of potent membrane-bound IL12 in adoptive cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB101.